AmpliTaq Gold™ DNA polymerase is a chemically modified enzyme that is rendered inactive until heated (Birch et al. 1996). An extended pre-incubation of 95°C, usually for 10 or 11 minutes, is used to activate the AmpliTaq Gold. The chemical modification involves a derivitization of the epsilon-amino groups of the lysine residues (Innis and Gelfand 1999). At a pH below 7.0 the chemical modification moieties fall off and the activity of the polymerase is restored. The Tris buffer in the PCR reaction is pH sensitive with temperature variation, and higher temperatures cause the solution pH to go down by approximately 0.02 pH units with every 1°C (Innis and Gelfand 1990). A Tris buffer with pH 8.3 at 25°C will go down to pH ~6.9 at 95°C. Thus, not only is the template DNA well denatured but the polymerase is activated just when it is needed, and not in a situation where primer dimers and mispriming can occur as easily.
It is important to note that AmpliTaq Gold is not compatible with the pH 9.0 buffers used for regular AmpliTaq DNA polymerases (Moretti et al. 1998). This fact is because the pH does not get low enough to remove the chemical modifications on TaqGold and thus the enzyme remains inactive. Tris buffers with a pH 8.0 or 8.3 at 25°C work the best with TaqGold.
Was this article helpful?
Discover Simple Techniques to Help Control Your Stutter. Stuttering is annoying and embarrassing. If you or a member of your family stutters, you already know the impact it can have on your everyday life. Stuttering interferes with communication, and can make social situations very difficult. It can even be harmful to your school or business life.